Antibiotic activities from streptosporangium vulgare ATCC 21906

ABSTRACT

Antibiotic activities produced by a variety of Streptosporangium vulgare named Streptosporangium vulgare var. antibioticum var. nov. ATCC 21906. The antibiotic complex contains at least the active fractions named A, B and C.

United States Patent Coronelli et al.

[451 Aug. 12, 1975 ANTIBIOTIC ACTIVITIES FROM STREPTOSPORANGIUM VULGARE ATCC lnventors: Carolina Coronelli; Maria Rosa Bardone; Hermes Pagani, all of Milan. Italy Assignee: Gruppo Lepetit S.p.A., Milan, ltaly Filed: May 24, 1974 Appl. No.: 473,023

Foreign Application Priority Data May 25, 1973 United Kingdom 25160/73 U.S. Cl. l95/80 R; 195/80 Int. Cl v t Cl2d 9/00 Field of Search l95/80 R [56] References Cited UNITED STATES PATENTS 2,990,325 6/1961 Dononick et al. 195/80 R Primary ExaminerA. Louis Monacell Assistant Examiner-Robert J. Warden Attorney, Agent, or FirmThe0d0re Post; C. Kenneth Bjork 3 Claims, No Drawings ANTIBIOTIC ACTIVITIES FROM STREPTOSIORANGIUM VULGARE ATCC 21906 SUMMARY OF THE INVENTION This invention relates to compositions of matter which have antibiotic properties and to methods for the manufacture and use thereof. The antibiotic activities of the invention are produced by a strain of Streptosporangium isolated from a soil sample collected in Bryanston (South Africa) and grown in submerged culture. On the basis of its morphological and physiological properties the new strain was considered a variety of Streplosporangium vulgare and was named Streptsp0- rangium vulgare var. antibioticum var. nor. A sample of this strain was deposited with the A.T.C.C. where it was assigned the number 21906. The antibiotic complex produced by Streptospurangium vulgare ATCC 21906 consists in at least three active fractions named A, B and C, which possess good in vitro and in vivo activity against Gram positive bacteria. Fraction C is endowed also with some antifungal activity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Characteristics of the producing strain To investigate the growth characteristics of strain Strepmsporangium vulgare ATCC 21906 the culture was grown on various standard media suggested by Shirling and Gottlieb Methods for characterization of Streptomyces species, Intern. J. Syst. Bact. 16: 313-338, l966) and on some media recommended by Waksman (The Actinomycetes, vol. II, The Williams and Wilkins Co., 1961). The morphological and cultural characteristics of Streptosporangium vulgare ATCC 2l906 in comparison with those of Streptosporangium vulgare CBS 43361 are reported in Table I. The optimum temperature for development was found to be from 28C to 37C and no growth was observed at C. The culture grows abundantly on oatmeal agar, Bennetts and I-Iickey-Tresners agar. On some culture media, the vegetative mycelium has coral-rose to orange color and the colonies are covered with an abundant rose colored aerial mycelium.

The tests for utilization of carbon compounds were performed according to Pridham and Gottlieb (The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bact. 56: 107, 1948.) and the results are shown in Table II. The physiological characteristics of the strain are described in Table III.

Cell wall composition of the new microorganism is shown in Table IV.

TABLE 1 Cultural characteristics of .S'lrepms xmuigimn rulgurt' \ur. umibiu/imm \ur. nor. ATCC 2l9llb in comparison with SIrt'pltIx mrungimn vulgurt' CBS/433.61.

(SMqsubstrate mycelium:

AM=aerial mycelium) MEDIUM .S'lrcplm' nmnigiuln rulgurc rur. SH'U/HUA'IKII'II!lgill! t'ulgurt' unlihiulit'um \UI'. or. ATCC 2l906 CBS/433.6]

Medium No. 2(Yeast extract- SM: abundant. wrinkled. orange to SM: abundant. wrinkled. amber malt agar) pink, light yellow pigment to vinaceous AM: abundant. powdery. pale pink AM: abundant. powdery. whitish Medium No. 3(()atmeal agar) SM: moderate, yellowish SM: moderate. yellowish AM: good. whitish AM: good, whitish Medium No. 4( Inorganic SM: poor. smooth and thin light SM: poor. smooth and thin. light salts-starch agar) yellow yellow AM: none AM: none Medium No. 5((ilycerol-aspa SM: poor. smooth surface. hyaline SM: poor. smooth surface. hyaline ragine agar) AM: none AM: none Medium No. (ilPeptone-yeast SM: poor. slightly wrinkled. amber SM: poor. slightly wrinkled extract iron agar) color amber color AM: none AM: none Medium No. 7(Tyrosine agar) SM: poor. slightly wrinkled. light SM: poor. slightly wrinkled.

brown light brown AM: none AM: none Oatmeal agar SM: good. wrinkled. pink-coral SM: go'od. wrinkled. pale pinkcoral AM: abundant. powdery. pink whitish AM: good. powdery. whitish Hickey and 'I'resner's agar SM: abundant. wn'nkled. light brown- SM: abundant. wrinkled. dark reddish brown-reddish AM: good. whitish AM: good. whitish Bennett's agar SM: abundant. wrinkled. cream color SM: abundant. wrinkled. light to pink amber-brown AM: abundant. pink-whitish AM: good. whitish C/apek glucose agar SM: poor. smooth and thin. hyaline SM: poor. smooth and thin.

amber colored AM: none AM: none (ilucose asparagine agar SM: poor. smooth. yellowish SM: poor. smooth. yellowish AM: none AM: none Nutrient agar SM: good. wrinkled surface. cream SM: good. wrinkled surface.

colored amber to vinaceous AM: none AM: poor. pinkish Potato agar SM: good. amber pinkish SM: good. amber colored AM: good. whitish AM: poor, whitish ('alcium malate agar SM: poor. smooth and flat. light SM: poor. smooth and flat.

yellow hyaline AM: none AM: none Skim milk agar SM: good. wrinkled. surface. deep SM: good. wrinkled surface.

orange deep orange AM: none AM: none Egg agar SM: \'cr scant. hyaline SM: \ery scant. hyaline AM: none AM: none Peptone glucose agar SM: poor. wrinkled. cream SM: poor. wrinkled. cream AM: nonc AM: none Agar SM: very scant. hyaline SM: very scant. hyaline AM none AM: traces. whitish TABLE 1 Continued Cultural characteristics of .S'IrcploS xlrulIgium \ulgul'r' \ur. unlihiulicum rur. Izm'. ATCC 21906 in comparison with .S'Irz'pmxpuru/ruin/u i'u/gurc CBS/433.61.

(SM=suhstrate mycelium;

MEDIUM Srrupms mrwzgium rulgurt' nu". untihiorit'um \ur. um. ATCC 2 1906 AM=aerial mycelium) 1 poor, hyaline to cream scant, cream none scant. cream none very scant. hyaline none scant. thin hyaline poor. whitish CBS (cntraalbureau Voor Schimmclcultures-Baarn (Netherlands) The number of the culture media refers to those given by Shirling and (iottlieb.

TABLE II Utilization of carbon sources by strain ATCC 21906 Carbon Source Response Inositol Fructose Rhamnose Mannitol Xylose Rafflnose Arabinose Cellulose Sucrose Glucose Mannose Lactose Salicin TABLE III Physiological properties of strain ATCC 21906 TABLE IV Cell-wall composition of strain ATCC/21906 Isomers of diamino- Amino Acids Sugars pimelic acid (DPA) Arabinose Xylose Galactose LL-DAP Meso or DD-DAP TR 11 Aspartic Glycine Lysine acid -11- major component minor component TR trace or doubtful component Analysis of cell-wall composition was performed according to the method described by B. Becker and H.

A. Lechevalier: Chemical composition of cell-wall preparations from strains of various form-genera of aerobic actinomycetes. Appl. Microbiol. 13:236-243. 1965.

At the microscopic examination the aerial mycelium of strain ATCC 21906 revealed to be formed of short highly branched hyphae, on the tip of which a spherical sporangium develops. The average diameter of the aerial mycelium is about 1.0a. The sporangia are quite variable in size, generally from 7 to 12 ,u. in diameter. The sporangiospores are disposed in a regular coiled fashion in the interior of the sporangium. The sporangiospores are generally spherical, non-motile, with an average diameter of 1.3 to l.5,u.. The non-motile sporangiospores are characteristic of the genus Streptosporangium according to the key for the genera of the family Actinoplanaceae and the cell-wall composition is in accordance.

A comparison of the cultural characteristics of our strain with those reported for the streptosporangia strains so far described showed a similarity with Streptosporangium vulgare(Nonomura and Ohara, Distribution of the Actinomycetes in soil. The isolation and classification of the genus Streptosporangium. J. Ferment. Technol. 38: 405, 1960), however the two strains can be differentiated for the color of the substrate and aerial mycelium on some media as shown by the data reported in Table I.

Since for Streptosporangium vulgare there is not described production of antibiotic activity the strain was assigned the name Streptusporangium vulgare var.antibioticum var. nov.

PRODUCTION AND ISOLATION OF THE ANTIBIOTIC yeast extract 1.0 g, soybean meal 1().() g. peptone 4.0 g. meat extract 4.0 g. dextrose 50.0 g. CaCQ- 5.0 g. NaCl 2.5 g.

1 liter tap water qs to The maximum antibiotic activity is obtained after 120-144 hours of fermentation. The microbiological assay is performed by the agar dissusion method using Staphylococcus aureus as the test organism.

For the extraction of the antibiotic activity the culture broth is filtered using Hyflo super-cell as a filter aid, the solution is treated with ethyl acetate, after addition of 1% sodium ascorbate. The solvent is washed with a phosphate buffer solution (pH 7.0) and concentrated in vacuo to a small volume. The resulting concentrate solution is poured into a large volume of light petroleum and a crude powder is obtained having an activity of 0.5y/ml against Staphylococcus aurcus.

BIOLOGICAL AND CHEMICO-PHYSICAL PROPERTIES OF THE CRUDE ANTIBIOTIC The crude mixture is active in vitro against grampositive bacteria at concentration values less than ly/ml and against gram-negative bacteria at concentration of about SO'y/mI. The crude complex is active at concentrations between 4 and 5'y/ml also against Slap/zylococcus aureus strains resistant to erythromycin and lincomycin.

Protection at low doses has been obtained against experimental infections in mice by subcutaneous route and at higher doses by oral route.

The antimicrobial spectrum and the results of experimental infections are reported in Table V and VI. The acute toxicity in mice is about 750 mg/kg.

The solubility of the crude mixture is good in alcohols, esters, chloroform and methylene chloride. The ultraviolet spectrum shows an absorption maximum at 260 mu for solutions at acidic pH and at 290 mu for solutions at alkaline pH. The pKa spectrophotometrically determined is about 5.5. The infrared spectrum shows intense peaks at 3200-3100 cm. and 1 100-1200 cm. characteristic for glycosidic moieties. Acid hydrolysis of the mixture gave substances with characteristic reactions and infrared spectra in accordance with a glycosidic nature of the antibiotic. A more detailed investigation has been carried out on the purifled fractions after separation by column chromatography.

PURIFICATION OF THE ANTIBIOTIC AND ANALYSIS OF THE SEPARATED FRACTIONS The presence of at least three active components has been revealed in the crude product by thin-layer chromatography using chloroform-methanol 95:5 or 98:2 mixtures as eluting system and microbiological development on Bacillus substilis as detecting system. Nicolaus et al., Il Farmaco Ed. Prat. 8, 350370, l96l A separation of the complex has been achieved by column chromatography on activated silica-infusorial earth (50:50 v/v) using chloroformmethanol mixture as eluting system. Three active fractions, named A, B and C, have been obtained, fraction C being still a mixture of two active components.

According to an effective procedure to carry out the separation, 6 g. of the crude complex are chromatographed on a column of 500 g. silica-infusorial earth 50:50 v/v) by eluting with fractions of 200 ml. Component A is eluted by a mixture CHCl -,:CH OH 98:2 evaporation of combined fractions 3 through 7 gives 0.655 g. of the product. Evaporation of the subsequent fractions 8 through 15 affords 810 mg. of a mixture of components A and B.

Component B is eluted by a mixture CHCI;,:CH OH :5 and is recovered from fraction 12 through 23. Evaporation of the combined eluates gives 1.1 g. of the pure component B.

Component C is eluted by a mixture CHCI :CH OH 90:10. Evaporation of the combined fractions 26 and 27 gives 0.935 g. of the product. As reported in Table IV the antibacterial spectra of fraction A and B are quite similar while fraction C shows some antifungal activity. Paper chromatography showed that only one of the two products contained in fraction C has antifungal properties.

The chemico-physical data reported in Table VI show a close relationship between the three fractions; the infrared spectra have the same general appearance and they all contain chlorine and nitrogen. From the microanalytical data of fractions A and B a minimum molecular formula containing two chlorine atoms and one nitrogen atom, with a molecular weight of about 1500, can be deduced. This is confirmed by the mass spectra of the two fractions, where the peaks at 630, 632 and 634 m/e, the highest ion peaks, have relative intensities in agreement with the presence of two chlorine atoms in this fragment. Also the determinations of the molecular weight through the dew point method support the value calculated from the chlorine content.

All three fractions A, B and C (l g.), when refluxed for 30 minutes in 0.1N hydrochloric acid ml.) gave after column chromatography of the methylene chloride extracts of each hydrolyzed mixture the same crystalline product, with m.p. l458C, [01],, (c=0.9% in pyridine for 24 hours):+ 30.9 17.6, A max in methanol at 279 mu, and microanalytical data in accordance for a molecular formula C H O Cl The aqueous phase from the hydrolysis after neutralization by anion exchange resin and evaporation in vacuo gave a solid residue positive to Fehling, ninhydrin, and red tetrazolium tests.

TABLE V In vitro spectrum of activity of the crude complex and fractions A. B and C ORGANISM TABLE V Continued In vitro spectrum of activity of the crude complex and fractions A. B and C ORGANISM CRUDE Minimal Inhibitory Concentration ('y/ml) COMPLEX FRACTION A FRACTION B FRACTION Slroplm'm'tux livnmLv/irus' C 203 0.05 0.05 0.01 0.1 Diplmm'cux pm'ullmliiut' NC 41 0.05 0.05 0.05 0.1 ('lm'lrillium JcI /i'iltuens' 155 30543 0.05 0.05 0.05 0.5 [:Ivc/uu-irhiu mli SK 12140 50 50 100 50 lrull'ux \ulgurix X 191 l ATCC 881 100 100 100 100 Rrmulmmumx ut'ruginmu ATCC 10145 100 100 100 100 M '0p/u.\'nm gullixvplicum H21 C.Z.B. Z 10 50 10 Mycu/uu'lt'rium mln'rculurix' H37, ATCC 9360 50 50 50 ('umlitlu uI/rimnx SKF 2270 20 100 100 'I'ric/mp/1 \'Iul| IlIt!lIlIjJHIp/[YH'S SKF 17410 50 100 100 10 TABLE V In vivo activity of the crude complex and fractions A. B and C (mg/kg] ORGANISM TREAT- CRUDE FRACTION A FRACTION B FRACTION MENT COMPLEX (ED-,0) C ROUTE ED,-,.,)

.S'lrz'plm'mt'ux /1('Il1t)l \[i('|l.\' C 203 st. 7.5 7.5 (ED 50 (ED,.,,) .S'Iuplrvlucm('11s llt'nmlvrit'us' C 203 no. 250 120 200 (ED,..,) Slap/ vimnu-x uurt'm' Tour s.c. 90 [)ipIm-m-cmr pm'unmniut' UC 41 s.c.

TABLE VI Chemico-physical properties of fractions A. B and C PROPERTIES FRACTION A FRACTION B FRACTION C RP 0 39 0.2 0.07 Microanalytical data C 51.8 C 52.2 Nitrogen and chlorine present H 6.3 H 6.4 No quantitative data reported N 0.9 N 0.95 because fraction C is a C I 4 3 C1 4.. mixture of two components.

IR. spectra 346W i )-1 720(m)- 1570(w)-1 150-1000(i) 630 highest ion peak 380 3460(i)172()(m)-165()(m) 1550(m)-1 15()-1()()0(i) 630 highest ion peak 380 233 base peak 233 base peak 233 base eak "Support: Silica gel (i/HF- plates. Solvent system chloroform:menthannl 98:2 10 X 2 cm). Antibiotics detected by development with concentrated H 50 at 100C.

"Infrared spectrum in nujol mull; intense peak i; medium peak in; weak peak w.

plcx therefrom and separating it into the three active fractions A, B and C.

2. Process as in claim 1 for producing the antibiotic complex where the fermentation is carried out at a temperature between about 25 and about 38C.

3. Process as in claim 1 where the separation of the antibiotic complex into the three active fractions is effected by column chromatography.

UNITED STATES PATENT OFFICE ERTEFICATE OF CORRECTION PATENTNQ: 3,899,396

DATED August 12, 1975 INVENTORt'S) C. Coronelli; Maria Rosa Bardone; H. Pagani it is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Table IV, colurrm 3, the headings are not over the right columns, correct as follows:

LL-DAP Meso or DD-DAP Aspartic Glycine Lysine Column 5, line 3, "dissusion" should read -diffusion;

Table VI, in footnote "chloroformmenthanol" should read -chloroform:methanol.

Signed and Sealed this fi D y of June1976 [SEAL] Arrest:

RUTH c. mason c. MARSHALL DANN H Commissioner oj'larems and Trademarks 

1. A PROCESS FOR PRODUCING ANTIBIOTIC ACTIVITY FROM STREPTOSPORANGIUM VULGARE VAR. ANTIBIOTICUM VAR. NOV. ATCC 21906 AND SEPARATING IT INTO THE THREE ACTIVE FRACTIONS NAMED A, B AND C WHICH COMPRISES CULTIVATING SAID MICROORGANISM UNDER SUBMERGED CONDITIONS IN A LIQUID NUTRIENT MEDIUM CONTAINING AN ASSIMILABLE SOURCE OF CARBON AND NITROGEN UNTIL A SUBSTANTIAL ANTIBIOTIC ACTIVITY IS PRESENT RECOVERING SAID ANTIBIOTIC COMPLEX THEREFROM AND SEPARATING IT INTO THE THREE ACTIVE FRACTIONS A, B AND C.
 2. Process as in claim 1 for producing the antibiotic complex where the fermentation is carried out at a temperature between about 25* and about 38*C.
 3. Process as in claim 1 where the separation of the antibiotic complex into the three active fractions is effected by column chromatography. 